Meningococcal polysaccharide conjugate vaccine

ABSTRACT

Neisseria meningitidis group B polysaccharide (GBMP) modified by having sialic acid residue N-acetyl groups replaced by N-acyl groups exhibits enhanced immuno response thereto. In addition, induction of antibodies which cross-react with unmodified group B meningococcal and E. coli K1 capsular polysaccharide and other tissue cells having a common epitope is minimized. Conjugation of the modified polysaccharides with a physiologically acceptable protein such as tetanus toxoid induces the production of specific protective antibodies with negligible levels of GBMP-cross reactive antibodies, to thereby afford protection against infections caused by group B meningococci and E. coli K1.

This is a divisional of application Ser. No. 08/238,600, filed May 5,1994, which is issued on Nov. 19, 1996 as U.S. Pat. No. 5,576,002, whichis a continuation of application Ser. No. 07/956,830, filed Oct. 5,1992, now abandoned, which is a continuation of application Ser. No.07/448,195, filed Dec. 14, 1989, now abandoned.

The present invention is directed to chemically-modified group Bpolysaccharides of Neisseria meningitidis. The invention also providesvaccines in which the respective modified polysaccharides are conjugatedto a protein carrier.

BACKGROUND OF THE INVENTION

Meningitis caused by group B N. meningitidis and E. coli K1 remain majorworld health problems. Group B meningitis occurs in both endemic andepidemic situations and accounts for approximately half of all recordedcases of meningococcal meningitis, while K1-positive E. coli are theleading cause of meningitis in neonates. Currently there is no vaccinecommercially available against disease caused by group B meningococciand E. coli K1. This is in large part due to the fact that the group Bmeningococcal polysaccharide (GBMP) is only poorly immunogenic inhumans. There are some recently reported candidate vaccines based oncomplexes of the GBMP with outer membrane proteins, but, as yet, thereis no clear evidence of their efficacy in humans.

Recently, a new concept of a vaccine based on a synthetic chemicallymodified (N-propionylated) group B polysaccharide-protein(N-Pr-GBMP-protein) conjugate has been developed. The vaccine induces inmice high titers of IgG antibodies which are not only protective, butalso cross-react with unmodified GBMP (i.e. N-acetyl-GBMP). This conceptis described and claimed in U.S. Pat. No. 4,727,136, issued Feb. 23,1988 to Harold J. Jennings et al.

It has been inferred that a vaccine which raises cross-reactiveantibodies such as that described in U.S. Pat. No. 4,727,136 could onlybe successful at the expense of breaking immune tolerance. Thishypothesis is legitimized by the identification of a common epitopeconsisting of a chain of α(2-8)-linked sialic acid residues (with aminimum requirement of ten residues) in both the native N-Ac-GBMP and inhuman and animal tissue (Jennings, Contrib. Microbiol. Immunol. Basel,Karger, 1989, Vol. 10, 151-165). These polysialosyl chains function asdevelopmental antigens and have for the most part been associated withthe fetal state in embryonic neural cell adhesion (Finne et al, Biochem.Biophys. Res. Commun., 1983, 112, 482). During post-natal maturation,this antigen is down-regulated (Friedlander et al, J. Cell Biol. 1985,101, 412) but is expressed in mature humans during the regeneration ofdiseased muscles (Cashman et al, Ann. Neuron., 1987, 21, 481) in tumorcells (Roth et al, Proc. Natl. Acad. Sci., 1988, 85, 299) and in naturalkiller (NK) and CD3⁺ T cells (Husmann et al, Eur. J. Immunol., 1989, 19,1761. Although the consequences of breaking tolerance to these fetalantigens have not yet been established, it is generally conceded that,because of this cross-reaction, the N-Pr-GBMP-protein conjugate would beseverely scrutinized by licensing agencies, resulting in considerableexpense and delays because of the complex experimentation necessary toprove the safety of the vaccine before its approval for commercialmarketing.

It is an object of the present invention to develop a vaccine havingimmunogenic properties which are enhanced as compared to those of theN-Pr-GBMP-protein. It is also an object of the invention to provide avaccine which exhibits substantially reduced cross-reactivity with GBMP.

SUMMARY OF THE INVENTION

In one aspect of the present invention, there is provided a modified Bpolysaccharide of Neisseria meningitidis having sialic acid residueN-acetyl (C₂) groups replaced by a C₄ -C₈ acyl group.

In another aspect, there is provided an antigenic conjugate comprisingthe N--C₄ -C₈ acyl polysaccharide conjugated to an immunologicallysuitable protein, having enhanced immunogenicity with substantiallyreduced inducement of cross-reactive antibodies.

In a further aspect, there is provided a vaccine comprising the N--C₄-C₈ acyl polysaccharide-protein conjugate in association with a suitablecarrier or diluent. The vaccines of the invention may also comprise atherapeutically effective amount of an adjuvant suitable for human use,for example aluminum phosphate or aluminum hydroxide.

In a yet further aspect, there is provided a method of immunizingmammals against N. meningitidis and E. coli K1 infections, which methodcomprises administering parenterally to mammals subject to suchinfections, including humans, an immunologically effective amount of thevaccine of the invention. The vaccine is typically administered in anamount of about 1 to 50 micrograms per kilogram body weight, for example5 to 25, micrograms per kilogram body weight.

In yet another aspect, the invention provides a gamma globulin fractioncapable of protection against meningitis caused by Group B Nmeningitidis and E. coli K1. The fraction is produced by immunizing amammal with a vaccine of the invention. The fraction is thenadministered to an individual to provide protection against or to treaton-going infection caused by the above organisms. From this, it will heappreciated that the immunogenic vaccine conjugates of the inventionwill provide for a source of therapeutic antiserum in light of theirfavorable immunogenicity with minimal inducement of GBMP cross-reactiveantibodies. The conjugates of the invention will also be useful forraising monoclonal antibodies and, possibly, antidiotype antibodies.

It has been found in our recent experiments that most of thebactericidal and protective antibodies induced by the N-Pr-GBMP-proteinconjugate described in the above-referred to Jennings et al U.S. Pat.No. 4,727,136 are not associated with the GBMP cross-reactiveantibodies. In fact, most of the protective activity is contained in anN-Pr-GBMP-specific antibody population which does not cross-react withGBMP. In light of this, it is believed that the N-Pr-GBMP mimics aunique bactericidal epitope on the surface of group B meningococci.

The present invention is based on the discovery that it is possible tosynthesize chemically modified GBMP's which mimic the bactericidalepitope and which, in their conjugated form, not only exhibit enhancedimmunogenicity but also avoid substantially the inducement of antibodiesthat cross-react with GBMP.

In arriving at the present invention, a number of different chemicallymodified GBMP's have been synthesized and conjugated individually toprotein, followed by injection of the conjugates into mice and theeffects compared to those produced by the N-Pr-GBMP protein conjugate.Surprisingly, it has been found that the N--C₄ -C₈ acyl GBMP-proteinconjugates, for example the n-butanoyl, iso-butanoyl, n-pentanoyl,iso-pentanoyl, neo-pentanoyl, hexanoyl, heptanoyl, and octanoyl, andespecially the N-butanoyl (N-Bu) GBMP-protein conjugate, substantiallymimic the bactericial epitope with substantially reduced inducement ofcross-reactive antibodies.

DETAILED DESCRIPTION OF THE INVENTION

The group B meningococcal polysaccharide is isolated from N.meningitidis by methods which are known in the art. In one such method,group B meningococci (strain 981B) were grown at 37° C. in a fermenterusing 30 g. of dehydrated Todd Hewitt Broth (Difco Laboratories,Detroit, Mich.) per liter of distilled water. Prior to fermenter growth,the lyophilized strain was grown initially in a candle jar at 37° C. on5% (v/v) Sheeps' Blood Agar (Difco Laboratories, Detroit, Mich.) plates.The bacteria were then transferred to 1.0 liter of Todd Hewitt Broth (asabove) in an Erlenmeyer flask which was shaken at 37° C. for 7 hours at190 r.p.m. This inoculum was then transferred to the fermenter. Afterfermenter growth (16 hours) the bacteria were killed by the addition offormalin to a final concentration of 0.75%. The bacteria were removed bycontinuous centrifugation and the group B meningococcal polysaccharidewas isolated from the supernatant and purified essentially as describedby Bundle et al, J. Biol. Chem., 249, 4797-4801 (1974) except that theprotein was extracted by stirring a solution of the crude polysaccharidewith cold (4° C.) 9% phenol instead of hot (50°-60° C). This latterprocess ensures that a high molecular weight form of the GBMP isproduced.

E. coli (018:K1:H7) (NRCC 4283) were grown at 37° C. in a fermenter indistilled water containing dehydrated Brain Heart Infusion (BHI; 37g/liter) (Difco Laboratories, Detroit, Mich.). Prior to fermentergrowth, the lyophilized strain was grown on 50 ml of BHI solution (sameas above) in an Erlenmeyer flask which was shaken at 37° C. for 7 hoursat 200 r.p.m. This growth was then transferred to 1.5 liters of BHI (asabove) and grown under the same conditions as described above for 7hours. The inoculum was then transferred to the fermenter.

The procedures employed in the isolation and purification of thecapsular polysaccharide of E. coli K1 were identical to those describedabove for the isolation of the group B meningococcal polysaccharide.

It will be appreciated that the isolation and purification proceduresdescribed above are not the only ones which may be utilized, and thatother published procedures are available, for example those described byWatson et al, J. Immunol., 81, 331 (1958) and in the above-mentionedU.S. Pat. No. 4,727,136.

The native polysaccharide is N-deacetylated to provide a reactive aminegroup in the sialic acid residue parts of the molecule. TheN-deacetylation can be carried out by any known method, for example in abasic aqueous medium at elevated temperatures, for example about 90° to110° C., and at a pH of about 13 to 14. The basic aqueous medium issuitably an aqueous alkali metal hydroxide solution, for example sodiumhydroxide of about 2M concentration. Alternatively, hydrazine in aqueoussolution may be used. The degree of N-deacetylation may vary from about30% to 100%, depending on the conditions. It is preferred to achieveabout 90 to 100% N-deacetylation. N-deacetylated product can berecovered for example by cooling, neutralizing, purification if desired,and lyophilization.

Prior to the N-deacetylation procedure, the native polysaccharide has anaverage molecular weight in the region of about 500,000 to 800,000Daltons. As a result of N-deacetylation, fragments of the polysaccharideare produced having an average molecular weight ranging from about10,000 to 50,000 Daltons.

The N-deacetylated polysaccharide fragments are then N-acylated toproduce the corresponding N-acylated product. The N-acylation may becarried out by dissolving the N-deacetylated polysaccharide in anaqueous medium having a pH of about 7.5 to 9.0, followed by adding theappropriate acyl anhydride, optionally with an alcohol to increasesolubility, and cooling to below 10° C. until the reaction is complete.The reaction medium can be purified, if desired, for example bydialysis, and the N-acylated product then recovered, typically bylyophilization. The reaction is substantially complete within about 10to 20 hours. The degree of N-acylation, as measured by analyticaltechniques, typically ¹ H nmr, is at least 90%, and likely close to100%. The N-acylation reaction does not result in any significantmolecular weight reduction of the fragments.

It is preferred, according to the present invention, to select, forconjugation purposes, the N-acylated material having an averagemolecular weight corresponding to about 30 to 200 sialic acid residues.This is generally achieved by way of gel filtration of the N-acylatedGBMP using an Ultragel (trademark) AcA 44 (Bead diameter 60-140 um)column, using PBS as eluant. Alternatively, a suitable sizing membranemay be employed.

N-acylated material of average molecular weight of 10,000 to 40,000Daltons, for example 10,000 to 15,000 Daltons, is employed for theinvention. This is obtained by collecting the fractions of the eluate ofthe column containing N-acylated GBMP material having that averagemolecular weight range. N-acylated material of higher average molecularweight, for example in the region of 30,000 to 40,000 Daltons, has alsoproved to be useful according to the invention.

The vaccines of the invention are produced by conjugating the N-acylatedpolysaccharide with an immunologically suitable carrier protein.Preferably, the carrier protein itself is an immunogen. Examples ofsuitable carrier proteins are tetanus toxoid, diphtheria toxoid,cross-reacting materials (CRMs), preferably CRM₁₉₇, obtained from SclavoLtd., Siena, Italy, and bacterial protein carriers, for examplemeningococcal outer membrane proteins.

Any mode of conjugation may be employed to conjugate the modifiedpolysaccharide fragments with the carrier protein. A preferred method isthat described in U.S. Pat. No. 4,356,170, i.e. by introducing terminalaldehyde groups (via oxidation of cis-vicinal hydroxyl groups) into theN-acylated polysaccharide and coupling the aldehyde groups to theprotein amino groups by reductive amination. The polysaccharide and theprotein are thereby linked through a --CH₂ --NH-protein linkage.

It is to be understood, however, that the conjugate vaccines of theinvention are not limited to those produced via reductive amination.Thus, the vaccines may also be produced by conjugating the N-acylatedpolysaccharide with the carrier protein using an adipic dihydrazidespacer, as described by Schneerson, R., et al, Preparation,Characterization and Immunogenicity of Haemophilus influenzae type bPolysaccharide-Protein Conjugates, J. Exp. Med., 1952, 361-476 (1980),and in U.S. Pat. No. 4,644,059 to Lance K. Gordon. Alternatively, theremay be used the binary spacer technology developed by Merck, asdescribed by Marburg, S., et al, "Biomolecular Chemistry ofMacromolecules: Synthesis of Bacterial Polysaccharide Conjugates withNeisseria meningitidis Membrane Protein", J. Am. Chem. Soc., 108,5282-5287 (1988) or, possibly, the reducing ends methodology, asreferred to by Anderson in U.S. Pat. No. 4,673,574.

The resulting N-acylated polysaccharide-protein conjugates do notpossess significant cross-linking and are soluble in aqueous solutions.This makes the conjugates of the invention good candidates for vaccineuse.

The resulting N-acylated-polysaccharide-protein conjugates of theinvention have been tested in vitro tests in mice, and have generallybeen shown to possess improved immunogenic properties as compared withthe N-propionylated-polysaccharide. In addition, substantially reducedformation of cross-reactive antibodies is observed. In light of this, itis believed that the vaccines of the invention will be useful againstmeningitis caused by group B N meningitidis or by E. coli K1 organisms.Of particular interest are vaccines for protecting human infants who aremost susceptible to bacterial meningitis.

The vaccines of the invention are typically formed by dispersing theconjugate in any suitable pharmaceutically acceptable carrier, such asphysiological saline or other injectable liquids. The vaccine isadministered parenterally, for example subcutaneously, intraperitoneallyor intramuscularly. Additives customary in vaccines may also be present,for example stabilizers such as lactose or sorbitol and adjuvants suchas aluminum phosphate, hydroxide, or sulphate.

A suitable dosage for the vaccine for human infants is generally withinthe range of about 5 to 25 micrograms, or about 1 to 10 micrograms perkilogram of body weight.

EXAMPLES

The invention is illustrated by the following non-limiting examples. TheN-acetyl, N-propionyl, N-butanoyl, N-isobutanoyl, N-pentanoyl andN-hexanoyl-GBMP-protein conjugates have been prepared for evaluationpurposes, and the results are discussed in the examples.

MATERIALS AND METHODS FOR PREPARING CONJUGATES

(a) Materials

Propionic, butanoic, isobutanoic, pentanoic, and hexanoic anhydridestogether with colominic acid were obtained from Sigma Chemicals Co., St.Louis, Mo. Because colominic acid is structurally identical to the groupB meningococcal polysaccharide (GBMP), it is referred to henceforth asGBMP. Tetanus toxoid (TT) was obtained from the institut ArmandFrappier, Laval, Quebec, and its monomeric form, used in all theconjugations, was obtained by passage of the above preparation through aBio-Gel (trademark) A 0.5 (200-400 mesh) column (1.6×90 cm) (Bio-Rad,Richmond, Calif.), equilibrated and eluted with 0.01M phosphate bufferedphysiologic saline (PBS) (pH 7.4)

(b) N-Deacetyation of the GBMP

The GBMP (Na⁺ salt) (1.0 g) was dissolved in 5 ml of 2M NaOH and,following the addition of NaBH₄ (150 mg), the solution was heated at110° C. for 6 hours in a screw cap Teflon (trademark) container (60 mL).This procedure is essentially as described in J. Immunol., 134, 2651(1985) and U.S. Pat. No. 4,727,136, both in the name of Harold J.Jennings, et al. The cooled diluted solution was then exhaustivelydialyzed against distilled water at 4° C., and lyophilized. The factthat N-deacetylated GBMP was obtained was determined by the absence ofthe methylacetamido signal (singlet at delta 2.07) in the ¹ H-nmrspectrum of the N-deacetylated GBMP.

(c) N-Acylations of the GBMP

N-Deacetylated GBMP (1.0 g) was dissolved in 50 mL of 5% aqueous NaHCO₃.To five individual aliquots (10 mL of the above solution) were addedeither propionic, butanoic, isobutanoic, pentanoic or hexanoicanhydrides. These reagents were added in 3×0.5 mL aliquots over a 3 hourperiod of time at room temperature while the solution was maintained atpH 8.0 with 0.5N NaOH. Methanol (0.5 mL) was added simultaneously witheach addition of anhydride in order to increase their solubility.Finally the solutions were stirred for 16 hours at 4° C., exhaustivelydialysed against distilled water at 4° C., and lyophilized. Theindividual N-propionylated, N-butanoylated, N-isobutanoylated,N-pentanoylated and N-hexanoylated GBMP were all obtained in yields inexcess of 90%. In each case, essentially complete N-acylation wasconfirmed by the disappearance in the respective ¹ H-nmr spectrum ofN-deacetylated GBMP.

(d) Sizing of the Fragments of the Different N-acylated GBMP

Gel filtration, using an Ultragel (trademark) AcA 44 (bead diameter60-140 μm) column (IBF Biotechnics, Savage, Md.) employed to obtain thedesired average molecular weight N-acylated GBMP material. Fractionseluting from the column at K_(D) 0.5 to K_(D) 0.7 as measured by FLPC(see below) were collected, dialyzed, and lyophilized. This range ofK_(D) values corresponds to N-acylated GBMP of approximately 30-50sialic acid residues (10,000 to 15,000 Daltons, typically 12,000 Daltonsaverage molecular weight). Fractions in the range of K_(D) 0.2 to 0.4corresponding to fragments having an average molecular weight in therange of 30,000 to 40,000 Daltons have also been collected andconjugated. Thus, N-acylated material eluting in the K_(D) range of 0.2to 0.7 is of particular interest.

(e) Polysaccharide Conjugates

Terminal aldehyde groups were introduced into the N-acylated GBMP byperiodate oxidation (see U.S. Pat. No. 4,356,170). The N-acylated GBMPfragments above were oxidized in 0.1M aqueous NaIO₄ (sodiummetaperiodate) (10 mL) for 2 hours at room temperature in the dark.Excess periodate was then destroyed by the addition of 1 mL of ethyleneglycol and the solution was then exhaustively dialyzed at 4° C, andlyophilized. The use of NaBH₄ in the N-deacetylation procedure (exceptfor the GBMP) results in the transformation of the terminal reducingsialic acid residues of each of the N-acylated GBMP, to open chainpolyol residues. This type of residue is periodate sensitive (see J.Immunol., 127, 1011 (1981) and U.S. Pat. No. 4,356,170 Harold J.Jennings et al), thereby resulting in the introduction of aldehydegroups into the N-acylated GBMP fragments at both termini.

The oxidized fragments (100 mg) were dissolved in 0.1M NaHCO₃ (pH 8.1)buffer (2 mL) and TT (20 mg) was added to the solution. Finally,following the addition of sodium cyanoborohydride (NaCNBH₃) (40 mg), thesolution was gently stirred at room temperature. The course of theconjugation was followed by FPLC using a gel filtration columncontaining SUPEROSE (trademark) 12 HR10/30 (Pharmacia), runisocractically at 1 mL/min in PBS buffer at pH 7.2, both the protein andN-acylated GBMP fragments being monitored at 214 nm. The fragments hadK_(D) 0.6, TT had K_(D) 0.39 and the conjugates had K_(D) 0.23. Theconjugation was complete when all the TT was expended as determined bythe loss of the peak in the FPLC chromatogram corresponding to thecomponent at K_(D) 0.39. In most cases, the conjugations were completein 2 days but were left for a total reaction time of 4 days. Thepotential unreacted aldehyde groups were finally reduced with sodiumborohydride (20 mg) prior to gel filtration.

The polysaccharide--TT conjugates were separated from the polysaccharidefragments by gel filtration using a Bio Gel A column with PBS as eluant.The eluant containing the conjugate was dialyzed against distilled waterand lyophilized. The N-acylated GBMP--TT conjugates contained from12-30%, typically 12-20%, sialic acid as determined by the resorcinolmethod described by Svennerholm, L., Quantitative Estimation of SialicAcids, II A Colorimetric Resorcinol-Hydrochloric Acid Method, Biochim.Biophys. Acta. 24, 604 (1957). This indicates that the conjugates had amolar ratio of polysaccharide to TT of 2-3:1 respectively.

IMMUNIZATION AND IMMUNOASSAYS

(a) Immunization Procedures

Twenty female white CFI mice (8-10 weeks old) were immunizedintraperitoneally (3 times at 3 week intervals) with each individualN-acylated GBMP-TT conjugate in Freunds' complete adjuvant (FCA) (Difco,Detroit, Mich.). Each immunization contained sufficient conjugate (10-12μg) to contain 2 μg of sialic acid. Eleven days after the thirdinjection, the mice were exsanguinated. The following tests were done onthe sera.

(b) Radioactive Antigen Binding Assay

This assay was carried out by a modification of the Fart technique usingextrinsically ³ H!-labeled GBMP (Jennings H. J., et al, DeterminantSpecificities of the Groups B and C polysaccharides of Neisseriameningitidis , J. Immunol., 134, 2651 (1985), or ³ H!-labeled N-Pr-GBMP(Jennings H. J., et al, Unique Intermolecular BaCtericidal Epitopeinvolving the Homo-Sialo Polysaccharide Capsule on the Cell Surface ofGroup B Neisseria meningitidis and Escherichia coli K1, J. Immunol.,142, 3585-3591 (1989). The reaction mixture for the radioactiveantigen-binding assay was obtained by mixing in Eppendorf polypropylenemicro test tubes 20 uL of pooled antisera, from groups of 20 miceimmunized with each individual N-acylated GBMP-TT conjugate, diluted to100 μL with PBS, with ³ H!-labeled GBMP and ³ H!-labeled N-Pr-GBMP in 50μL of PBS. After incubation at 4° C. for 16 hours, 150 μL of saturated(at 4° C.) ammonium sulfate (pH 7.0) was added to the tubes and thetubes agitated and left to stand at 4° C. for 30 min. The tubes werecentrifuged at 15,000 rpm for 10 min. and two aliquots of 130 μL weredrawn from the tubes. The aliquots were mixed with 2 mL of water and ascintillant-containing xylene (ACS aqueous scintillant) and the mixtureswere counted in a liquid scintillation counter. Results are given inTable 1.

                  TABLE 1                                                         ______________________________________                                        labeled - N--Ac--GBMP to different                                            mouse anti- N--acyl--GBMP--TT conjugate sera.                                                % Binding.sup.a                                                Antiserum        1     2         3   4                                        ______________________________________                                        N--Pr--GBMP--TT  41    40        39  12                                       N--Bu--GBMP--TT   4     4         7   4                                       N--IsoBu--GBMP--TT                                                                              9    --        --  --                                       N--Pen--GBMP--TT 36    --        --  --                                       N--Hex--GBMP--TT 16    --        --  --                                       ______________________________________                                         .sup.a The four binding experiments were carried out on pooled antisera       from 20 immunized mice.                                                       Abbreviations used in Table 1 and other tables: N--Ac--, N--Pr, N--Bu,        N--IsoBu, N--Pen, N--Hex-- stand for N--Acetyl, N--Propionyl--,               N--Butanoyl--, N--Isobutanoyl--, N--Pentanoyl-- and N--Hexanoyl--.       

The numerals 1, 2, 3 and 4 are results of four repeat experiments. Table1 demonstrates conclusively that the N-Ac-GBMP (which carries the sameepitope as fetal N-CAM) binds less to the antiserum induced by theN-Bu-GBMP, N-IsoBu-GBMP, N-Pen-GBMP and N-Hex-GBMP than that induced bythe N-Pr-GBMP. From this, it can be deduced from Table 1 that the N-Bu-,N-IsoBu, N-Pen- and N-Hex-polysaccharide-conjugates raise lesscross-reactive antibodies than the N-Pr-conjugate.

(c) Quantitative Precipitin Analyses

These experiments were carried out by the method of Kabat and Mayer,Experimental Immunochemistry Charles C. Thomas, Springfield, p.22(1961). Aliquots (100 μL) of anti-N-acyl GBMP-TT sera (diluted 5 fold inPBS) were reacted in tubes with increasing concentrations of theN-acetyl (colominic acid), N-propionyl, N-butanoyl, N-isobutanoyl,N-pentanoyl and N-hexanoyl GBMP in a total volume of 200 μL (adjustedwith PBS). The higher molecular weight fractions of these derivativeswere used in these experiments and they were obtained from the eluate ofthe Ultragel AcA 44 column (K_(D) 0.4 as measured by FPLC) previouslyused to size the fragments of the N-acylated GBMP. The tubes wereincubated at 4° C. for 4 days with daily mixing, centrifuged, and thequantity of antibody protein was determined by the method of Lowry etal, Protein Measurement with the Folin phenol reagent, J. Biol. Chem.,1933, 265 (1951). The results are given in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Precipitation.sup.a of mouse anti-N--acyl--GBMP--TT sera                      using different N--acyl GBMP as precipitating antigens.                                   N--acyl--GBMP antigen                                             Antiserum   N--acetyl                                                                          N--propyl                                                                          N--butyl                                                                           N--pentyl                                                                          N--hexyl                                      __________________________________________________________________________    N--Pr--GBMP--TT                                                                           0.16 0.40 0.20 0.15 0.15                                          N--Bu--GBMP--TT                                                                           0.04 1.15 2.60 3.20 1.90                                          N--Pen--GBMP--TT                                                                          0.13 0.38 0.44 6.35 3.55                                          N--Hex--GBMP--TT                                                                          0.02 0.08 0.80 4.15 4.40                                          __________________________________________________________________________     .sup.a Maximum amount of antibody precipitated expressed in mg/mL of          antiserum                                                                

As regards cross-reactivity, the first column of Table 2 indicates thatvery little cross-reactive antibodies are produced by the N-Bu and N-Hexconjugates as compared to the N-Pr conjugate. It can also be seen thatthe N-Pen conjugate produces less cross-reactive antibodies than theN-Pr conjugate.

With reference to immunogenicity, in terms of homologous response, itcan be seen from Table 2 that the N-Bu- (2.60), N-Pen- (6.35) and N-Hex-(4.40) GBMP-TT conjugates are more immunogenic than the N-Pr-GBMP analog(0.40).

(d) ELISA

The wells of EIA microtitration plates (Flow Labs, Mississauga, Ontario,Canada) were coated with a 10 μg/mL solution of either GBMP-, NPrGBMP-or NBu-GBMP-BSA conjugates in PBS (100 μL/well). The plates were leftfor 18 hours at 4° C. and after coating they were washed with 1% bovineserum albumin in PBS for 10 min. at room temperature (blocking step).The wells were then filled with 100 μL of serial 10-fold dilutions inPBS of anti-mouse-N-acyl GBMP-TT conjugate sera and the plates wereincubated for 1 hour at room temperature. After washing with SBT theplates were incubated for 1 hour at room temperature with 50 μL of theappropriate dilution of goat antimouse immunoglobulin peroxidase labeledconjugates (Kirkegard and Perry Laboratories), then the contents of thewells were aspirated and the plates washed five times with SBT. Finally50 μL of Tetramethylene blue-peroxidase substrate (TMB) (Kirkegard andPerry Laboratories) were added to each well after 10 min the absorbanceat 450 nm was measured with a Multiscan spectrophotometer (FlowLaboratories, Mississauga, Ont.). Results are given in Table 3.

                                      TABLE 3                                     __________________________________________________________________________    ELISA titrations of pooled mouse anti-N--acyl--GBMP--TT conjugate             serum against N--acyl--GBMP--BSA conjugates.                                  Coating     Titers.sup.a of Antisera                                          Antigen     a. N--Pr--GBMP.sup.b                                                                   a. N--Bu--GBMP.sup.b                                                                   a. N--Isobu--GBMP.sup.b                         __________________________________________________________________________    N--Ac--GBMP--BSA                                                                           7800     1000    7000                                            N--Pr--GBMP--BSA                                                                          40000    39000    9800                                            N--Bu--GBMP--BSA                                                                          26000    52000    9700                                            N--IsoBu--GBMP--BSA                                                                       --       --       25000                                           __________________________________________________________________________     .sup.a titer (GM) = reciprocal of dilution at 50% of the maximum              absorbance at 450 nm.                                                         .sup.b N--acyl specific antisera induced in mice by homologous                N--acyl--GBMP--TT conjugates.                                            

With reference to cross-reactivity, it can be seen from Table 3 that theN-Bu-GBMP-TT conjugate raises less cross-reactive antibodies withrespect to N-Ac-GBMP (1000) than does the N-Bu-GBMP-TT conjugate (7800).The reason for this is that the GBMP binds less to antibody induced bythe N-Pr-GBMP-TT conjugate than that induced by the N-Pr-GBMP-TTconjugate. Similar comments apply with respect to the N-IsoBut-GBMP-TTconjugate.

As regards immunogenicity, the N-Bu conjugate is more immunogenic thanthe N-Pt analogue, as shown by the homologous binding titers of 52,000(N-Bu) and 40,000 (N-Pr).

(e) Radioactive Binding Inhibition Assay

Increasing concentration of the larger molecular sized N-acyl GBMPinhibitor in PBS (80 μL) were added to 20 μL of mouse anti-N-Pr-GBMP-TTconjugate antiserum, an amount sufficient to bind 50% of the (³H)-labeled N-Pr-GBMP in the absence of inhibitor. The tubes wereincubated for 1 hour at 37° C. and 50 μL of (³ H)-labeled N-Pr-GBMP inPBS was added. After gentle mixing the tubes were incubated at 4° C. for16 hours and the assays were performed exactly as described previouslyfor the radioactive antigen binding assay. The % inhibition wascalculated using the following formula:

percent inhibition=100× (cpm with inhibitor minus cpm withoutinhibitor)/(cpm without antibody minus cpm without inhibitor)!.

Results are given in Table 4.

                  TABLE 4                                                         ______________________________________                                        labeled N--Pr--GBMP to mouseup.3 H!                                           anti-N--Pr--GBMP--TT conjugate induced IgG.sub.2a, IgG.sub.2b (A).sup.a       and IgG.sub.1 (B).sup.a antibodies.                                           Inhibitor.sup.b    A      B                                                   ______________________________________                                        N--Ac--GBMP        >50.0  >50.0                                               N--Pr--GBMP        0.6    0.3                                                 N--Bu--GBMP        0.3    0.2                                                 N--IsoBu--GBMP     >50.0  --                                                  N--Pen--GBMP       2.3    2.5                                                 N--Hex--GBMP       10.2   10.0                                                ______________________________________                                         .sup.a These were fractions of mouse polyclonal antiN--Pr--GBMP--TT,          serum, described in Jennings et al, J. Immunol., 142, 3585-3591 (1989).       .sup.b Micrograms of inhibitor to give 50% inhibition.                   

Bactericidal Assays

These assays were carried out by the method described by Jennings etal., J. Exp. Med., 165, 1207-1211 (1987).

Neisseria meningitidis strain B (M 986) was used in these assays.Results are given in Table 5.

                  TABLE 5                                                         ______________________________________                                        labeled - N--Pr--GBMP to different mouse                                      anti-N--acyl--GBMP--TT conjugate sera and the bactericidal                    titers of the respective antisera.                                            Antiserum     μL of antiserum.sup.a                                                                   Bactericidal Titer.sup.b                           ______________________________________                                        N--Pr--GBMP--TT                                                                             13           128                                                N--Bu--GBMP--TT                                                                             10           64                                                 N--IsoBu--GBMP--TT                                                                          ND           64                                                 N--Pen--GBMP--TT                                                                            24           64                                                 N--Hex--GBMP--TT                                                                            >100          8                                                 N--Ac--GBMP--TT                                                                             >100          8                                                 ______________________________________                                         .sup.a μL of antiserum (diluted 5 fold with PBS) required for 50%          binding.                                                                      .sup.b Dilution experiment: one dilution difference, e.g. 128 compared to     64, is within experimental error.                                        

Table 4 illustrates that N-Bu-GBMP is as good an inhibitor as theN-Pr-GBMP for the binding of the latter to its own homologousantibodies. Therefore, the use of N-Bu-GBMP in place of N-Pr-GBMP doesnot result in any significant loss of properties exhibited by N-Pr-GBMP.Table 5 demonstrates that the N-Bu-GBMP binds as well as the N-Pr-GBMPto antibodies induced by the N-Pr-GBMP-TT conjugate. Antisera induced byboth the N-Bu-GBMP-TT and N-Pr-GBMP-TT conjugates gave similarbactericidal titers. This evidence indicates the equivalence of theN-Bu-, N-IsoBu- and N-Pen-GBMP to the N-Pr-GBMP in their ability tomimic the bactericidal epitope on the surface of the group Bmeningococci.

We claim:
 1. A gamma globulin fraction comprising antibodies directedagainst group B N. meningitidis or E. coli K1, said gamma globulinfraction being produced by immunizing a mammal with an antigenicconjugate comprising a modified group B polysaccharide of Neisseriameningitidis or E. coli K1 having sialic acid N-acetyl residue groupsreplaced by a N--C4-C₈ -acyl group conjugated to animmunologically-suitable protein, wherein the the N-acetyl groups arereplaced with the N--C₄ -C₈ acyl groups to an extent that when saidmodified group B polysaccharide is conjugated to said protein the amountof substitution of acyl for acetyl groups is sufficient to reduce theinducement of antibodies which bind native GBMP, compared to the bindingof native GBMP to antibodies induced by a N-pr-GBMP-proteinconjugate--has been inserted.
 2. The gamma globulin fraction accordingto claim 1, wherein the C₄ -C₈ acyl group of the conjugate used toimmunize the mammal is selected from the group consisting of n-butanoyl,isbutanoyl, n-pentanoyl, n-hexyanol, n-heptanoyl and n-octanoyl.
 3. Thegamma globulin fraction according to claim 1, wherein the C₄ -C₈ acylgroup of the conjugate used to immunize the mammal is selected from thegroup consisting of n-butanoyl, isbutanoyl, n-pentanoyl and n-hexyanoyl.4. The gamma globulin fraction according to 3, wherein the C₄ -C₈ acylgroup of the conjugate used to immunize the mammal is n-butanoyl.
 5. Thegamma globulin fraction according to claim 4 wherein the protein of theconjugate elicits antibodies to bacteria.
 6. The gamma globulin fractionaccording to claim 5 wherein the protein of the conjugate is selectedfrom the group consisting of tetanus toxoid, diphtheria toxoid,cross-reacting material (CRM) and a meningococcal outer membraneprotein.
 7. The gamma globulin fraction according to claim 6 wherein theprotein and the polysaccharide of the conjugate are covalently linkedthrough a --CH₂ --NH-- linkage and wherein the N of the linkage iscontributed by the protein.
 8. The gamma globulin fraction according toclaim 7 wherein the C₄ -C₈ acyl group of the conjugate is of n-butanoyl.9. The gamma globulin fraction according to claim 8, wherein the CRM isCRM₁₉₇.